Etanercept Formulations Stabilized with Sodium Chloride

ABSTRACT

The invention provides stabilized aqueous pharmaceutical etanercept compositions suitable for long-term storage of etanercept, methods of manufacture of these compositions, methods of administration, and kits containing same.

FIELD OF THE INVENTION

The present invention relates to aqueous pharmaceutical compositionsstabilized with sodium chloride for long-term storage of etanercept,methods of manufacture of the compositions, methods of theiradministration, and kits containing the same. The invention includesetanercept formulations that do not require arginine for stabilization.

BACKGROUND OF THE INVENTION

Polypeptides must often be stored prior to their use. When stored forextended periods, polypeptides are frequently unstable in solution(Manning et al., 1989, Pharm. Res. 6:903-918). To extend their shelflife, additional processing steps have been developed, such as drying,e.g., lyophilization. However, lyophilized pharmaceutical compositionsare less convenient to use.

Typical practices to improve polypeptide stability can be addressed byvarying the concentration of elements with the formulation, or by addingexcipients to modify the formulation (See, for example, U.S. Pat. Nos.5,580,856 and 6,171,586). However, the use of additives can still resultin inactive polypeptides. In addition, in the case of lyophilization,the rehydration step can result in inactivation of the polypeptide by,for example, aggregation or denaturation (Flora et al., 1992, Pharm.Res., 9:33-36; Liu et al., 1991, Biotechnol. Bioeng., 37:177-184).Aggregation of polypeptides is undesirable, as it may result inimmunogenicity (Cleland et al., 1993, Crit. Rev. Therapeutic DrugCarrier Systems, 10:307-377; and Robbins et al., 1987, Diabetes,36:838-845).

Another way to improve polypeptide stability is to use L-arginine at aspecific concentration (U.S. Pat. No. 7,648,702).

One of the polypeptides that is stored for up to two years prior to useis etanercept (Enbrel®, Immunex Corporation), which is a dimeric fusionpolypeptide consisting of the extracellular ligand-binding portion ofthe human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR)linked to the Fc portion of human IgG1. It consists of 934 amino acidsand has an apparent molecular weight of approximately 150 kilodaltons(Physicians Desk Reference, 2002, Medical Economics Company Inc.) The Fccomponent of etanercept contains the constant heavy 2 (CH2) domain, theconstant heavy 3 (CH3) domain and hinge region, but not the constantheavy 1 (CH1) domain of human IgG1. An Fc domain can contain one or allof the domains described above. Etanercept is usually produced byrecombinant DNA technology in a Chinese hamster ovary (CHO) mammaliancell expression system.

The present invention provides novel stable liquid formulations ofetanercept that allow its long-term storage.

SUMMARY OF THE INVENTION

The present invention is an aqueous etanercept formulation stabilized toreduce instability, aggregation and/or fragmentation of the etanercept,said formulation comprising about 25 to about 75 mg/ml of etanercept andone or more stabilizers, wherein the stabilizers are selected from thegroup consisting of (i) sodium chloride; and (ii) sodium chloride incombination with sucrose or trehalose; and (iii) a combination of sodiumchloride, sucrose and trehalose.

Various technical terms used in the following discussion are definedbelow in the section entitled “Definitions” and throughout the remainderof the specification.

The stabilized etanercept formulations of the present invention elicitlong term storage stability as characterized by at least one of: (1) SECanalysis at M₃ or T₂ or T₄ of: monomer content greater than about 90%;aggregates content of less than about 3 wt %; and fragment 3 contentless than about 5 wt %: and (2) HIC analysis at M₃ or T₂, or T₄ whereinthe amount of the composition represented by peak 1 of the HICchromatogram is less than about 3 wt. %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than 80 wt. %;and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 20 wt. %.

In a related aspect the formulations elicit long term storage stabilityas characterized by: (1) SEC analysis at M₃ or T₂ or T₄ of greater thanabout 90 wt. % monomer content; less than about 3 wt. % aggregate(s)content; and less than about 5 wt % fragment 3; and (2) HIC analysis atM₃ or T₂ or T₄ wherein the amount of the composition represented by peak1 of the HIC chromatogram is less than about 3 wt. %; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan 80 wt. %; and the amount of the composition represented by peak 3of the HIC chromatogram is less than about 20 wt. %; and wherein theformulation is free or substantially free of arginine.

In preferred aspects of the stabilized formulations, the formulationselicit long term storage stability as characterized by: an HIC analysisat M₃ or T₂ or T₄ wherein the amount of the composition represented bypeak 2 of the HIC chromatogram is greater than or equal to about 95 wt.%; and wherein, if peak 3 is present on the HIC chromatogram, the amountof the composition represented by peak 3 is less than or equal to about3 wt. %.

The stabilized etanercept formulation as summarized above, optionallyand preferably, contains no arginine, or are essentially free ofarginine.

The formulations of the invention have excellent stability as determinedby SEC (Size Exclusion Chromatography) and HIC (Hydrophobic InteractionChromatography) analysis conducted after one, two or three months ofstorage at 5° C. These formulations are comparable to or better than acommercially available formulation of etanercept, in which arginine is arequired component. Accordingly the present invention is furtherdirected to formulations of stabilized etanercept, as summarized above,which contain no arginine, or are essentially free of arginine, andwherein the composition at M₃ or T₂ or T₄ elicits long term storagestability that meets one or both of the following criteria: (A)stability comparable to or better than commercially available etanerceptmarketed under the trademark Enbrel®, as measured by (i) SEC analysis ofthe amounts of aggregate(s), monomer and fragment 3 in the composition(as defined in the specification) and (ii) HIC analysis of amounts ofmaterial in the composition corresponding to peaks 1, 2 and 3 of the HICchromatogram (as defined in the specification); and (B) an HICchromatogram in which (i) peak 3 is absent, or essentially absent and(ii) peak 2 represents greater than about 95 wt % of the composition; anSEC chromatogram containing essentially no peak corresponding toaggregate(s); and an SEC chromatogram in which the monomer contentrepresents at least about 95 wt % of the composition.

In one preferred aspect, the formulation of the invention comprisesabout 25 to 75 mg/ml etanercept, up to about 150 mM of sodium chloride,about 1 to about 30 mM sodium phosphate; and about 0 to 5 wt % sucroseor trehalose or combination thereof; wherein the composition has a pH ofabout 6.0 to about 6.6; and wherein the composition is characterized bySEC analysis at M₃ or T₂ or T₄ in which: monomer content is greater thanabout 80 wt. %; aggregate(s) content is less than about 3 wt %, andfragment 3 content is less than about 8 wt. %. Moreover, formulationsmeeting these criteria do not require arginine for stabilization.

In a further preferred embodiment the stabilized etanercept formulationis further characterized by (a) an SEC analysis at M₃ or T₂ or T₄ ofgreater than about 90 wt % monomer content; and less than about 3 wt %aggregate(s) content; and (b) an HIC analysis at M₃ or T₂ or T₄ whereinthe amount of the composition represented by peak 1 of the HICchromatogram is less than about 4 wt %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than about 80wt %; and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 20 wt %. Formulations meeting thesecriteria do not require arginine for stabilization.

The etanercept compositions of the invention further afford the abilityto provide formulations which contain acceptable levels of subvisibleparticles. Accordingly, the invention is further directed to sodiumchloride stabilized etanercept formulations having, at M₃ or T₂ or T₄ nomore than, on average, about 10,000 subvisible particles per mL having asize greater than 5 μm.

The stabilized etanercept composition of the present invention arefurther characterized by: (a) an SEC analysis at M₃ or T₂ or T₄ ofgreater than about 90 wt % monomer content; and less than about 3 wt %aggregate(s) content; and (b) an HIC analysis at M₃ or T₂ or T₄ whereinthe amount of the composition represented by peak 1 of the HICchromatogram is less than about 3 wt %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than 80 wt %;and the amount of the composition represented by peak 3 of the HICchromatogram is less than about 20 wt %. Formulations meeting thesecriteria do not require arginine for stabilization.

The stability of the formulations may be further characterized in thatthe compositions exhibit an HIC analysis at M₃ or T₂ or T₄ wherein theamount of the composition represented by peak 1 of the HIC chromatogramis less than about 1%; the amount of the composition represented by peak2 of the HIC chromatogram is greater than about 95 wt %; and the amountof the composition represented by peak 3 of the HIC chromatogram is lessthan about 1 wt %. Surprisingly, formulations meetring these criteria donot require arginine for stabilization.

In a further preferred embodiment, the sodium chloride stabilizedetanercept formulation of the invention comprises up to about 150 mMsodium chloride, about 1 to 30 mM sodium phosphate, and about 0-5 wt. %sucrose or trehalose, or combination of sucrose and trehalose and havinga pH of about 6.0 to 6.6; and characterized by: an SEC analysis at M₃ orT₂ or T₄ of greater than about 95 wt. % monomer content and less thanabout 1 wt % aggregate(s) content; and an HIC analysis at M₃ or T₂ or T₄wherein the amount of the composition represented by peak 1 of the HICchromatogram is less than or equal to about 3 wt %; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan about 82 wt %; and the amount of the composition represented bypeak 3 of the HIC chromatogram is less than about 15 wt %. Preferably,formulations meeting these analytical criteria do not contain, or aresubstantially free of arginine.

It is surprising and unexpected that excellent stabilization can beprovided in an etanercept formulation without need for arginine as astabilizing component. Accordingly the present invention is directed tothe following sodium chloride stabilized formulations which contain noarginine or are substantially free of arginine:

-   -   An etanercept formulation free of arginine, comprising: about 50        mg/ml of etanercept; about 75 mM sodium chloride; about 10 mM        sodium phosphate; about 3 wt. % sucrose.    -   An etanercept formulation free of arginine, comprising: about 50        mg/ml of etanercept; about 75 mM sodium chloride; about 25 mM        sodium phosphate; about 3 wt. % trehalose.    -   An etanercept formulation free of arginine, comprising: about 50        mg/ml of etanercept; about 75 mM sodium chloride; about 25 mM        sodium phosphate; about 3 wt. % sucrose and having pH of about        6.3 to 6.5.    -   An etanercept formulation free of arginine, comprising: about 50        mg/ml of etanercept; about 150 mM sodium chloride; about 25 mM        sodium phosphate, in the absence of sucrose or trehalose.    -   An etanercept formulation free of arginine, comprising: about 50        mg/ml of etanercept; about 100 mM sodium chloride; about 15 mM        sodium phosphate; and about 2 wt. % sucrose.

Preferred stabilized compositions of the invention, preferably free ofarginine, exhibit an HIC analysis at M₃ or T₂ or T₄ wherein the amountof the composition represented by peak 1 of the HIC chromatogram is lessthan about 2% or preferably less than about 1%; the amount of thecomposition represented by peak 2 of the HIC chromatogram is greaterthan about 95 wt. % and preferably greater than about 97%; and theamount of the composition represented by peak 3 of the HIC chromatogramis less than about 1 wt %, and preferably 0 to 1%.

As differentiated from commercially available etanercept provided in anarginine-containing formulation, we found it surprising, in light ofU.S. Pat. No. 7,648,702, that the formulation embodiments of etanerceptdescribed and exemplified herein do not require arginine for long-termstabilization, although arginine may still be added if desired. Theability to provide etanercept formulations stabilized without argininerepresents a potentially significant benefit to the health care systemby providing patients and health care providers with alternativeformulations of etanercept that may become available at lower costcompared with present commercial etanercept formulation (i.e., Enbrel®)that require arginine for stabilization.

As used herein the term “instability” or like terms denotes the tendencyof the etanercept monomer to undergo a variety of undesiredtransformations during storage. Such transformations include theformation of oligomers and high molecular weight aggregate(s)(hereinafter terms “aggregate(s)” in which multiple copies of theessentially intact etanercept monomer become irreversibly associatedwith one another through a variety of non-covalent attractions (e.g.,electrostatic interactions.) Undesired transformations during storagemay also include degradation of the etanercept monomer to smallerfragments and/or clipped species. Ideally, a formulation of etanerceptshould minimize, to the greatest extent possible, the tendency of theformulation to result, during storage, in the formation of aggregates,misfolded protein, oligomers and/or fragments of etanercept. Animportant benefit resulting from the ability to reduce formation ofunwanted aggregates or fragments is a reduction in the potentialtoxicity and/or immunogenicity of the drug.

The etanercept formulation of the present invention which is optionallyand preferably free, or essentially free of arginine. The term“essentially free of arginine” is intended to mean that arginine, evenif present, is not contributing to the stabilization of the etanerceptmonomer in the formulation to such an extent that a person skilled inthe art would judge its presence beneficial or necessary from astabilization standpoint.

These and other aspects will become apparent from the followingdescription although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are now described in detail. Asused in the description and throughout the claims, the meaning of “a”,“an”, and “the” includes plural reference unless the context clearlydictates otherwise. Also, as used in the description and throughout theclaims, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise. Additionally, some terms used in thisspecification are more specifically defined below.

DEFINITIONS

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. Synonyms for certain termsare provided. A recital of one or more synonyms does not exclude the useof other synonyms. The use of examples anywhere in this specificationincluding examples of any terms discussed herein is illustrative only,and in no way limits the scope and meaning of the invention or of anyexemplified term. The invention is not limited to the variousembodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

“Around,” “about” or “approximately” shall generally mean within 20percent, within 10 percent, within 5, 4, 3, 2 or 1 percent of a givenvalue or range. Numerical quantities given are approximate, meaning thatthe term “around,” “about” or “approximately” can be inferred if notexpressly stated.

The term “etanercept” or “etanercept monomer” or “monomer” is synonymouswith Enbrel®. It refers to a polypeptide which is a dimeric fusionpolypeptide consisting of the extracellular ligand-binding portion ofthe human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR)linked to the Fc portion of human IgG1. It consists of 934 amino acidsand has an apparent molecular weight of approximately 150 kilodaltons.For the purposes of the present application, the term “etanercept” alsoencompasses etanercept with minor modifications in the amino acidstructure (including deletions, additions, and/or substitutions of aminoacids) which do not significantly affect the function, potency, oravidity of etanercept. The term “etanercept” encompasses all forms andformulations of Enbrel®, including but not limited to concentratedformulations, injectable ready-to-use formulations; formulationsreconstituted with water, alcohol, and/or other ingredients, and others.

The term “sugar” refers to monosaccharides, disaccharides, andpolysaccharides. Examples of sugars include, but are not limited to,sucrose, trehalose, dextrose, and others.

The term “polyol” refers to an alcohol containing multiple hydroxylgroups. Examples of polyols include, but are not limited to, mannitol,sorbitol, and others.

The term “long-term storage” is understood to mean that thepharmaceutical composition can be stored for three months or more, forsix months or more, and preferably for one year or more. Long-termstorage is also understood to mean that the pharmaceutical compositionis stored either as a liquid at 2-8° C., or is frozen, e.g., at −20° C.,or colder. It is also contemplated that the composition can be frozenand thawed more than once.

The term “stable” or “stabilized” with respect to long-term storage isunderstood to mean that etanercept contained in the pharmaceuticalcompositions does not lose more than 20%, or more preferably 15%, oreven more preferably 10%, and most preferably 5% of its activityrelative to activity of the composition at the beginning of storage.

The term “mammal” includes, but is not limited to, a human.

The term “pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material,formulation auxiliary, or excipient of any conventional type. Apharmaceutically acceptable carrier is non-toxic to recipients at thedosages and concentrations employed and is compatible with otheringredients of the formulation.

The term “composition” refers to a mixture that usually contains acarrier, such as a pharmaceutically acceptable carrier or excipient thatis conventional in the art and which is suitable for administration intoa subject for therapeutic, diagnostic, or prophylactic purposes. It mayinclude a cell culture in which the polypeptide or polynucleotide ispresent in the cells or in the culture medium. For example, compositionsfor oral administration can form solutions, suspensions, tablets, pills,capsules, sustained release formulations, oral rinses or powders.

The terms “pharmaceutical composition” and “formulation” are usedinterchangeably.

The term “treatment” refers to any administration or application ofremedies for disease in a mammal and includes inhibiting the disease,arresting its development, relieving the disease, for example, bycausing regression, or restoring or repairing a lost, missing, ordefective function; or stimulating an inefficient process. The termincludes obtaining a desired pharmacologic and/or physiologic effect,covering any treatment of a pathological condition or disorder in amammal. The effect may be prophylactic in terms of completely orpartially preventing a disorder or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disorder and/oradverse affect attributable to the disorder. It includes (1) preventingthe disorder from occurring or recurring in a subject who may bepredisposed to the disorder but is not yet symptomatic, (2) inhibitingthe disorder, such as arresting its development, (3) stopping orterminating the disorder or at least its associated symptoms, so thatthe host no longer suffers from the disorder or its symptoms, such ascausing regression of the disorder or its symptoms, for example, byrestoring or repairing a lost, missing or defective function, orstimulating an inefficient process, or (4) relieving, alleviating orameliorating the disorder, or symptoms associated therewith, whereameliorating is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, such as inflammation, pain and/or tumorsize.

The term “disease” refers to any condition, infection, disorder orsyndrome that requires medical intervention or for which medicalintervention is desirable. Such medical intervention can includetreatment, diagnosis and/or prevention.

The term “therapeutically effective amount” refers to an amount which,when administered to a living subject, achieves a desired effect on theliving subject. For example, an effective amount of the polypeptide ofthe invention for administration to the living subject is an amount thatprevents and/or treats an integrin αvβ3-mediated disease. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art, adjustments for systemic versus localized delivery,age, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art.

The term “T₁” refers to a point in time at which an etanerceptformulation has been stored for about one week at 40° C.

The term “T₂” refers to a point in time at which an etanerceptformulation has been stored for about two weeks at 40° C.

The term “T₄” refers to a point in time at which an etanerceptformulation has been stored for about four weeks at 40° C.

The term “M₃” refers, collectively, to three points in time, and inparticular to an analytical result being observed for an etanerceptformulation after duration of either about one, about two or about threemonths of storage at a storage temperature of 5° C. For example,reference herein to an analysis being conducted at M₃ should beunderstand to mean that such analysis is be done at the point in time atwhich etanercept formulation has been in storage for a duration selectedfrom about one, about two, or about three months. Thus, a requirementherein that an etanercept formulation elicit a certain analytical valueor measurement at M₃ is satisfied if the required value is observed at apoint in time corresponding to at least one of the following storagedurations: at approximately one month, at approximately two months or atapproximately three months of storage at 5° C.

The terms “Peak 1,” Peak 2″ and “Peak 3” when used herein in connectionwith discussion of HIC chromatography results refers to the same peaks1, 2 and 3 discussed in U.S. Pat. No. 7,294,481.

EMBODIMENTS OF THE INVENTION

When pharmaceutical compositions containing etanercept (Enbrel®),including aqueous and lyophilized formulations of etanercept are storedon a long term basis, the activity of etanercept can be lost ordecreased due to instability of the etanercept monomer via aggregationand/or chemical degradation including formation of fragments. Thus, thepresent invention provides several embodiments of aqueous formulationsof etanercept that allow stable long-term storage of etanercept, so thatetanercept is stable over the course of storage either in liquid orfrozen states. The provided formulations include, but are not limited toformulations that do not contain arginine and do not require any extrasteps such as rehydrating.

These embodiments are explained in a greater detail below.

Etanercept

All of the compositions of the present invention comprise etanercept(Enbrel®). As explained in the Background section of this application,etanercept is a dimeric fusion polypeptide consisting of theextracellular ligand-binding portion of the human 75 kilodalton (p75)tumor necrosis factor receptor (TNFR) linked to the Fc portion of humanIgG1. Etanercept consists of 934 amino acids. The Fc component ofetanercept contains the constant heavy 2 (CH2) domain, the constantheavy 3 (CH3) domain and hinge region of human IgG1. An Fc domain cancontain one or all of the domains described above.

Etanercept suitable for storage in the present pharmaceuticalcomposition can be produced by living host cells that expressetanercept, such as hybridomas in the case of antibodies, or host cellsthat that have been genetically engineered to produce the polypeptide inthe case of fusion polypeptides or antibodies. Methods of geneticallyengineering cells to produce polypeptides are well known in the art.See, e.g., Ausubel et al., eds. (1990), Current Protocols in MolecularBiology (Wiley, New York). Such methods include introducing nucleicacids that encode and allow expression of the polypeptide into livinghost cells. These host cells can be bacterial cells, fungal cells, or,preferably, animal cells grown in culture. Bacterial host cells include,but are not limited to, Escherichia coli cells. Examples of suitable E.coli strains include: HB101, DH5.alpha, GM2929, JM109, KW251, NM538,NM539, and any E. coli strain that fails to cleave foreign DNA. Fungalhost cells that can be used include, but are not limited to,Saccharomyces cerevisiae, Pichia pastoris and Aspergillus cells. A fewexamples of animal cell lines that can be used are CHO, VERO, BHK, HeLa,Cos, MDCK, 293, 3T3, and W138. New animal cell lines can be establishedusing methods well know by those skilled in the art (e.g., bytransformation, viral infection, and/or selection). Optionally,etanercept can be secreted by the host cells into the medium.

Purification of the expressed etanercept can be performed by anystandard method. When etanercept is produced intracellularly, theparticulate debris is removed, for example, by centrifugation orultrafiltration. When etanercept is secreted into the medium,supernatants from such expression systems can be first concentratedusing standard polypeptide concentration filters. Protease inhibitorscan also be added to inhibit proteolysis and antibiotics can be includedto prevent the growth of microorganisms.

Etanercept can be purified using, for example, hydroxyapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography, and any combination of known or yet to be discoveredpurification techniques, including but not limited to Protein Achromatography, fractionation on an ion-exchange column, ethanolprecipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSET®, an anion or cation exchange resinchromatography (such as a polyaspartic acid column), chromatofocusing,SDS-PAGE, and ammonium sulfate precipitation.

Etanercept Formulations Stabilized with NaCl

The invention provides an aqueous etanercept formulation stabilized toreduce instability, aggregation and/or fragmentation of the etanercept,said formulation comprising about 25 to about 75 mg/ml of etanercept andone or more stabilizers, wherein the stabilizers are selected from thegroup consisting of (i) sodium chloride and (ii) sodium chloride incombination with sucrose or trehalose; and (iii) a combination of sodiumchloride, sucrose and trehalose.

The pharmaceutical compositions of the invention may be prepared bycombining, a purified etanercept and sodium chloride, optionally withsucrose and/or trehalose. Further, a buffer, a tonicity modifier and anadditional excipient and other commonly used inactive ingredients can beadded as needed. For simplicity, these are discussed more fully later inthe specification. A person of ordinary skill in the art will understandthat the combining of the various components to be included in thecomposition can be done in any appropriate order. For example, thebuffer can be added first, middle or last, and the tonicity modifier canalso be added first, middle or last. A person of ordinary skill in theart will also understand that some of these chemicals can beincompatible in certain combinations, and accordingly, are easilysubstituted with different chemicals that have similar properties butare compatible in the relevant mixture.

In an embodiment using sodium chloride for stabilization, an etanerceptformulation of the invention comprises about 25 to 75 mg/ml etanercept,up to about 150 mM of sodium chloride, about 1 to about 30 mM sodiumphosphate; and about 0 to 5 wt. % sucrose or trehalose or combinationthereof; wherein the composition has a pH of about 6.0 to about pH 7.0,and more preferably about 6.0 to about 6.6 and most preferably about 6.3to about 6.5.

The sodium chloride stabilized composition is preferably characterizedby SEC analysis at T₂ in which: monomer content is greater than about 80wt. %; aggregate(s) content is less than about 3 wt. %, and fragment 3content is less than about 8 wt. %.

The sodium chloride-stabilized etanercept composition is preferablycharacterized by:

-   -   (a) an SEC analysis at T₄ of greater than about 90, 91, 92, 93,        94, 95, 96, or 97 wt. % monomer content; and less than about 3,        2 or 1 wt. % aggregate(s) content; and    -   (b) an HIC analysis at T₂ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        4, 3, 2 or 1 wt. %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 85 or 86 wt. %; and the amount of the composition represented        by peak 3 of the HIC chromatogram is less than about 20, 19, 18,        17, 16, 15, 14, or 13 wt. %; and    -   (c) an HIC analysis at T₄ wherein the amount of the composition        represented by peak 1 of the HIC chromatogram is less than about        3, 2 or 1 wt. %; the amount of the composition represented by        peak 2 of the HIC chromatogram is greater than 80, 81, 82, 83,        84 or 85 wt. %; and the amount of the composition represented by        peak 3 of the HIC chromatogram is less than 20, 19, 18, 17, 16,        15, 14 or 13 wt. %.

In a further embodiment, preferred composition using sodium chloride forstabilization comprise up to about 150 mM sodium chloride, about 1 to 30mM sodium phosphate, and about 0-5 wt. % sucrose or trehalose, orcombination of sucrose and trehalose and having a pH of about 6.0 to6.6; and characterized by: an SEC analysis at T₄ of greater than about95 wt. % monomer content and less than about 1 wt. % aggregate(s)content; an HIC analysis at T₂ wherein the amount of the compositionrepresented by peak 1 of the HIC chromatogram is less than or equal toabout 3 wt. %; the amount of the composition represented by peak 2 ofthe HIC chromatogram is greater than about 82 wt. %; and the amount ofthe composition represented by peak 3 of the HIC chromatogram is lessthan about 15 wt. %; and an HIC analysis at T₄ wherein the amount of thecomposition represented by peak 1 of the HIC chromatogram is less thanabout 2 wt. %; the amount of the composition represented by peak 2 ofthe HIC chromatogram is greater than about 84 wt. %; and the amount ofthe composition represented by peak 3 of the HIC chromatogram is lessthan or equal to about 14 wt. %.

Particularly preferred compositions in terms of reduced aggregates andfragments are those in which the sodium chloride stabilized etanerceptformulations exhibit HIC analysis at T₄ or T₂ wherein the amount of thecomposition represented by peak 1 of the HIC chromatogram is less thanabout 1%; the amount of the composition represented by peak 2 of the HICchromatogram is greater than about 95 wt. % and preferably greater thanabout 99 wt. %; and the amount of the composition represented by peak 3of the HIC chromatogram is less than about 3 wt. %.

In a further embodiment of the invention, an NaCl stabilized etanerceptformulation contains up to about 5 mM arginine.

In the above-referenced NaCl stabilized etanercept formulations, theterms “SEC”, “T₂” “T₄” “HIC” “monomer content” “aggregate(s)” and“fragment 3” “peak 1,” “peak 2,” and “peak 3,” are defined in theexamples below.

Additional Components of the Provided Pharmaceutical Compositions

The formulations of the invention may also include buffers, tonicitymodifiers, excipients, pharmaceutically acceptable carriers and othercommonly used inactive ingredients of the pharmaceutical compositions.For simplicity, these are discussed more fully later in the application.

Buffers maintain pH in a desired range. Suitable buffers includehistidine, potassium phosphate, sodium or potassium citrate, maleicacid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris),various forms of acetate and diethanolamine. The concentration of thebuffer in the formulation is preferably between about 1 mM to about 1M,and more preferably about 10 mM to about 200 mM. Buffers are well knownin the art and are manufactured by known methods and available fromcommercial suppliers.

Examples of suitable buffers are phosphate, histidine, citrate, maleate,tartrate, succinate, acetate, tris-(hydroxymethyl)-aminomethane (tris),bicarbonate.

In a preferred embodiment, the buffer is sodium phosphate.

In a preferred embodiment, the pH of the pharmaceutical composition isat or near physiological levels. Thus, preferably, the pH of theprovided compositions is between about 5.8 and about 8.4; and even morepreferably, between about 6.2 and about 7.4. A person of ordinary skillin the art will understand that the pH can be adjusted as necessary tomaximize stability and solubility of etanercept in a particularformulation. Thus, etanercept formulations at a pH outside ofphysiological ranges, yet tolerable to the patient, are also within thescope of the invention.

A tonicity modifier is a molecule that contributes to the osmolality ofa solution. The osmolality of a pharmaceutical composition is preferablyadjusted to maximize the active ingredient's stability and/or tominimize discomfort to the patient upon administration. It is generallypreferred that a pharmaceutical composition be isotonic with serum,i.e., having the same or similar osmolality, which is achieved byaddition of a tonicity modifier.

In a preferred embodiment, the osmolality of the provided formulationsis from about 180 to about 420 mOsM. However, it is to be understoodthat the osmolality can be either higher or lower as specific conditionsrequire.

Examples of tonicity modifiers suitable for modifying osmolalityinclude, but are not limited to amino acids (not including arginine)(e.g., cysteine, histidine and glycine), salts (e.g., sodium sulfate,potassium chloride and sodium citrate) and/or saccharides (e.g.,sucrose, glucose and mannitol).

Preferred tonicity modifiers are glycine, alanine, sodium chloride,potassium chloride, and sodium sulfate.

In a preferred embodiment, the concentration of the tonicity modifier inthe formulation is preferably between about 1 mM to about 1 M, morepreferably about 10 mM to about 200 mM. Tonicity modifiers are wellknown in the art and are manufactured by known methods and availablefrom commercial suppliers.

Excipients, also referred to as chemical additives, co-solutes, orco-solvents, that stabilize the polypeptide while in solution (also indried or frozen forms) can also be added to a pharmaceuticalcomposition. Excipients are well known in the art and are manufacturedby known methods and available from commercial suppliers.

Examples of suitable excipients include but are not limited tosugars/polyols such as: sucrose, lactose, glycerol, xylitol, sorbitol,mannitol, maltose, inositol, trehalose, glucose; polymers such as: serumalbumin (bovine serum albumin (BSA), human SA or recombinant HA),dextran, poly(viny alcohol) PVA, hydroxypropyl methylcellulose (HPMC),polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP),hydroxyethylcellulose (HEC); non-aqueous solvents such as: polyhydricalcohols, (e.g., PEG, and glycerol) and dimethylformamide (DMF); aminoacids such as:

proline, L-serine, sodium glutamic acid, alanine, glycine, lysinehydrochloride, sarcosine and gamma-aminobutyric acid; surfactants suchas: Tween®-80 (polysorbate 80), Tween®-20 (polysorbate 20), SDS,polysorbate, poloxamers; and miscellaneous excipients such as: potassiumphosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodiumsulfate, trimethylamine N-oxide, betaine, metal ions (e.g., zinc,calcium, and magnesium), CHAPS, monolaurate, 2-O-beta-mannoglycerate orany combination of the above.

Preferred excipients are sucrose, lactose, glycerol, xylitol, sorbitol,mannitol, maltose, inositol, trehalose, glucose, bovine serum albumin(BSA), human serum albumin (HSA), recombinant albumin, dextran, PVA,hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin,polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), polyethyleneglycol, ethylene glycol, glycerol, alanine, glycine, lysinehydrochloride, sarcosine, SDS, polysorbate 20, polysorbate 80, poloxamer188, trimethylamine N-oxide, betaine, zinc ions, calcium ions, magnesiumions, CHAPS, sucrose monolaurate, and 2-O-beta-mannoglycerate.

The concentration of one or more excipients in a formulation of theinvention is/are preferably between about 0.001 to 5 weight percent,more preferably about 0.1 to 2 weight percent.

Methods of Treatment

In another embodiment, the invention provides a method of treating amammal comprising administering a therapeutically effective amount ofthe pharmaceutical compositions of the invention to a mammal, whereinthe mammal has a disease or disorder that can be beneficially treatedwith etanercept.

In a preferred embodiment, the etanercept is derived from the samespecies of mammal as is to be treated with the composition.

In a preferred embodiment, the mammal is a human.

Diseases or disorders that can be treated with the provided compositionsinclude but are not limited to rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Wegener's disease (granulomatosis),Crohn's disease (or inflammatory bowel disease), chronic obstructivepulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia,psoriasis, and atopic dermatitis. Additional diseases or disorders thatcan be treated with the compositions of the present invention includethose described in WO 00/62790, WO 01/62272, U.S. Patent Application No.2001/0021380, and U.S. Pat. No. 7,648,702 B2, the relevant portions ofwhich are incorporated herein by reference.

The provided pharmaceutical compositions may be administered to asubject in need of treatment by injection systemically, such as byintravenous injection; or by injection or application to the relevantsite, such as by direct injection, or direct application to the sitewhen the site is exposed in surgery; or by topical application.

In one embodiment, the invention provides a method of treatment and/orprevention of rheumatoid arthritis comprises administering to a mammalin need thereof a therapeutically effective amount of one of theprovided etanercept compositions.

The therapeutically effective amount of the etanercept in the providedcompositions will depend on the condition to be treated, the severity ofthe condition, prior therapy, and the patient's clinical history andresponse to the therapeutic agent. The proper dose can be adjustedaccording to the judgment of the attending physician such that it can beadministered to the patient one time or over a series ofadministrations.

In one embodiment, the effective etanercept amount per adult dose isfrom about 1-500 mg/m², or from about 1-200 mg/m², or from about 1-40mg/m² or about 5-25 mg/m².

Alternatively, a flat dose may be administered, whose amount may rangefrom 2-500 mg/dose, 2-100 mg/dose or from about 10-80 mg/dose.

If the dose is to be administered more than one time per week, anexemplary dose range is the same as the foregoing described dose rangesor lower and preferably administered two or more times per week at a perdose range of 25-100 mg/dose.

In another embodiment, an acceptable dose for administration byinjection contains 80-100 mg/dose, or alternatively, containing 80 mgper dose.

The dose can be administered weekly, biweekly, or separated by severalweeks (for example 2 to 8).

In one embodiment, etanercept is administered at 25 to 75 mg/ml by asingle subcutaneous (SC) injection.

In some instances, an improvement in a patient's condition will beobtained by administering a dose of up to about 100 mg of thepharmaceutical composition one to three times per week over a period ofat least three weeks. Treatment for longer periods may be necessary toinduce the desired degree of improvement. For incurable chronicconditions the regimen may be continued indefinitely. For pediatricpatients (ages 4-17), a suitable regimen may involve administering adose of 0.4 mg/kg to 5 mg/kg of etanercept, one or more times per week.

In another embodiment, the pharmaceutical formulations of the inventionmay be prepared in a bulk formulation, and as such, the components ofthe pharmaceutical composition are adjusted to be higher than would berequired for administration and diluted appropriately prior toadministration.

The pharmaceutical compositions can be administered as a soletherapeutic or in combination with additional therapies as needed. Thus,in one embodiment, the provided methods of treatment and/or preventionare used in combination with administering a therapeutically effectiveamount of another active agent. The other active agent may beadministered before, during, or after administering the pharmaceuticalcompositions of the present invention. Another active agent may beadministered either as a part of the provided compositions, oralternatively, as a separate formulation.

Administration of the provided pharmaceutical compositions can beachieved in various ways, including parenteral, peroral, buccal,sublingual, nasal, rectal, intraperitoneal, intradermal, transdermal,subcutaneous, intravenous, intra-arterial, intracardiac,intraventricular, intracranial, intratracheal, intrathecaladministration, intramuscular injection, intravitreal injection, andtopical application.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, i.e., subcutaneously,intramuscularly, intravenously, intraperitoneal, intracerebrospinal,intra-articular, intrasynovial, intravitreal, and/or intrathecal.Parenteral administration can be by bolus injection or continuousinfusion. Pharmaceutical compositions for injection may be presented inunit dosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. In addition, a number of recent drug deliveryapproaches have been developed and the pharmaceutical compositions ofthe present invention are suitable for administration using these newmethods, e.g., Inject-ease®, Genject®, injector pens such as GenPen®,and needleless devices such as MediJector® and BioJector®. The presentpharmaceutical composition can also be adapted for yet to be discoveredadministration methods. See also Langer, 1990, Science, 249:1527-1533.

The provided pharmaceutical compositions can also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the formulations may bemodified with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions may, if desired, be presented in a vial,pack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. In one embodiment the dispenser devicecan comprise a syringe having a single dose of the liquid formulationready for injection. The syringe can be accompanied by instructions foradministration.

In another embodiment, the present invention is directed to a kit orcontainer, which contains an aqueous pharmaceutical composition of theinvention. The concentration of the polypeptide in the aqueouspharmaceutical composition can vary over a wide range, but is generallywithin the range of from about 0.05 to about 20,000 micrograms permilliliter (μg/ml) of aqueous formulation. The kit can also beaccompanied by instructions for use.

The present invention is more particularly described in the followingexamples that are intended as illustrative only, since manymodifications and variations therein will be apparent to those skilledin the art.

Example Etanercept Stabilized with NaCl

Etanercept formulations stabilized with NaCl, alone, or NaCl incombination with sucrose, trehalose and/or arginine, may be prepared andtested using the procedures generally described below.

Each solid formulation component is weighed to the amount required for agiven volume of formulation buffer. These components are combined into abeaker or vessel capable of carrying and measuring the given volume offormulation buffer. A volume of deionized water equal to approximately ¾of the target given formulation buffer is added to the beaker, and thecomponents are then solublized. The pH of the buffer is adjusted to thetarget formulation pH using 1 M sodium hydroxide and/or 1 M hydrogenchloride. The final formulation buffer volume is then raised to thetarget volume through the addition of deionized water. Etanerceptprotein solution is placed in dialysis material housing (such as ThermoScientific Slide-A-Lyzer MINI Dialysis Unit 10,000 MWCO), which is thenplaced in contact with the desired formulation buffer for 12 hours at 4°C. Formulation buffer volume to protein solution volume ratio should beno less than 1000:1. The dialysis housing and protein solution itcontains is then placed in a second, equal volume of formulation bufferfor an additional 12 hours at 4° C. Resulting protein solution isremoved from the dialysis material housing, and the concentration ofprotein determined using ultraviolet spectroscopy. Protein concentrationis adjusted to the desired level using centrifugation (such as AmiconUltra 10,000 MWCO Centrifugal Concentrators) and/or dilution withformulation buffer.

The compositions can be tested for long-term stability by size exclusionchromatography (SEC), denatured SEC (dSEC), hydrophobic interactionchromatography (HIC), sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE), and for binding and bioactivity at varioustimepoints. The bioactivity can be measured by any number of well-knownassays.

For example, the techniques of Size Exclusion Chromatography aredescribed in Hawe et al, Pharm. Res. 2011, 28: 2302 and/or vanMarrschalkerweerd et al., Eur. J. Pharm. Biopharm. 2011, 78: 213.Similarly, the techniques of Denatured Size Exclusion Chromatography,Hydrophobic Interaction Chromatography, and SodiumDodecylSulfate-PolyAcrylamide Gel Electrophoresis are also well known topersons having ordinary skill in the art.

It is believed that the composition will be stable over the term of twoyears or more.

(Formulation 2:8) Ingredient concentration Etanercept (activeingredient) 50 mg/ml Sodium phosphate, pH 6.32 (inactive) 25 mM NaCl(inactive) 150 mM

(Formulation 2:6) Ingredient concentration Etanercept (activeingredient) 50 mg/ml Sodium phosphate, pH 6.3 (inactive) 15 mM NaCl(inactive) 100 mM Sucrose (inactive) 2% (w/v)

(Formulation 3:10) Ingredient concentration Etanercept (activeingredient) 50 mg/ml Sodium phosphate, pH 6.57 (inactive) 10 mM NaCl(inactive) 75 mM Sucrose (inactive) 3% (w/v)

(Formulation 3:11) Ingredient concentration Etanercept (activeingredient) 50 mg/ml Sodium phosphate, pH 6.30 (inactive) 25 mM NaCl(inactive) 75 mM Trehalose (inactive) 3% (w/v)

(Formulation 3:12) Ingredient concentration Etanercept (activeingredient) 50 mg/ml Sodium phosphate, pH 6.3 (inactive) 25 mM NaCI(inactive) 75 mM Sucrose (inactive) 3% (w/v)

(Formulation 3:13) Ingredient concentration Etanercept (activeingredient) 50 mg/ml Sodium phosphate, pH 6.3 (inactive) 25 mM NaCl(inactive) 120 mM Sucrose (inactive) 1% (w/v) Arginine (inactive) 5 mM

Example 2 Preparation of Etanercept

Step 1.

Cell Expansion.

In a manner known in the art, cell expansion necessary to generate asufficient number of cells for inoculation of a production bioreactor isperformed using a clone of CHO cells expressing the etanercept fusionprotein. The product of this expression process (a harvested cellculture fluid) results in a mixture of correctly folded etanercept, aswell as incorrectly folded and/or aggregated etanercept, along withadditional impurities. The harvested cell culture fluid comprising suchprotein mixture is subjected to detergent viral inactivation.

Step 2.

Affinity Chromatography.

Affinity chromatography is performed on the harvested cell cultureobtain in Step 1 above using a conventional Protein A affinity column ina well known manner. Product recovery is approximately 85%. The productobtained is a complex protein mixture comprising correctly foldedetanercept, incorrectly folded etanercept, and/or aggregates ofcorrectly and/or incorrectly folded etanercept, or protein fragments.The product obtained from this Protein A affinity column purificationstep is adjusted to pH 3.5 and then subjected to a viral inactivationstep. Following viral inactivation the product is adjusted to pH 5.5 andthen further clarified in a known manner using a commercially obtainedcapsule filter.

Step 3A.

Mixed-Mode Cation Exchange Chromatography.

A 31.8 L (45 cm diameter×20 cm bed height) packed bed GE HealthcareCapto MMC chromatography column is used to purify the product obtainedin Step 2 above. Prior to use, the column is equilibrated with 2 CV of25 mM acetate pH 5.5 and sanitized with 2 CV of 0.1 N NaOH, 1 M NaCl andneutralized with 2 CV of 25 mM acetate, 0.7 M NaCl, pH 5.5. The columnis then equilibrated with 8-10 CV of 25 mM acetate pH 5.5 until theeffluent is pH 5.5 and 3.5 mS/cm. The Protein A pool from step 2 aboveis diluted to ≦6 mS/cm with WFI and applied to a column loading of up to15 g/L media for each cycle. The column is operated at a linear velocityof 200 cm/h to give a 6 minute residence time. After loading, the columnis washed with 2 CV of 25 mM acetate pH 5.5. The product is then elutedwith an 8.5 CV, 15% to 85% gradient of 25 mM acetate pH 5.5 to 25 mMacetate, 0.7 M NaCl, pH 5.5. Product collection begins at 0.15 OD (A280,1.0 cm path length) and collection ends at 50% of peak maximum. Theeluate volume is approximately 5 CV. Residual product and contaminantsare stripped from the column with 2 CV of 10 mM Tris, 1 M NaCl, pH 8.0and discarded. The product obtained from the mixed mode column isfiltered using a Millipore Opticap XL10, 0.22 μm Durapore capsulefilter, (0.69 m²). The product obtained from this step represents arecovery of about 70% of the Protein A material obtained in Step 2

Step 3B.

Mixed Mode Anion Exchange Chromatopgraphy.

A 27.0 L (45 cm diameter×17 cm bed height) packed bed GE HealthcareCapto Adhere chromatography column is used to further purify the productobtained in step 3A above. Prior to use, the column is equilibrated with2 CV of 25 mM Tris, pH 8.0 and sanitized with 2 CV of 0.1 N NaOH, 1MNaCl and neutralized and equilibrated with 2 CV of 25 mM Tris, pH 8.0.Prior to product loading, the column is equilibrated with 3 CV of 10 mMTris, pH 8.0. The Capto MMC pool from Step 3A above is adjusted to pH8.1 with ˜0.045 kg of 1 M Tris, pH 8.3 per kg of pool. The product fromStep 3A above was diluted in-line 1:3.8 with WFI to adjust theconductivity to 12.0 mS/cm and pH 8.0. The resulting material is thenapplied to a column loading of up to 15 g/L media. The column isoperated at a linear velocity of 170 cm/h to give a 6 minute residencetime. After loading, the column is washed with 2 CV of 25 mM Tris, pH8.0. The product is then eluted with a 10 CV gradient (20% to 90%) of 25mM Tris, pH 8.0 to 10 mM Tris, 1 M NaCl, pH 8.0. Product collection isstarted at 0.15 OD (A280, 1.0 cm path length) and collection ended at25% of peak maximum. The eluate volume is 4-6 CV. The eluted product isfiltered using a commercially available capsule filter and thensubjected in a known manner to viral filtration and tangential flowfiltration steps. Overall product recovery from step 3B (including thefinal viral and tangential flow filtration steps) was approximately 68%.Product recovery measured before the filtration steps was about 75%. Aschematic representation of HIC data obtained on eluation fractions fromthis step are representing in FIG. 12.

Analysis:

The final filtered product obtained in this example is found to havegreater than about 90 wt % correctly folded etanercept as determined byHIC; less than 5 wt % incorrectly folded etanercept species asdetermined by HIC; less than about 3 wt % of clipped material by HICanalysis (believed to be fragments of etanercept in which the TNFRportion thereof has been truncated) and a combined amount of correctlyand incorrectly folded etanercept of greater than 95 wt % as determinedby size exclusion chromatography.

Analysis of Etanercept Formulations

A. Thermal Stability Storage

Following dialysis and concentration, samples of the etanerceptformulations exemplified above were sterile filtered in a bio safetycabinet. Using sterilized pipettes and autoclaved pipette tips, samplesof the etanercept formulations were transferred to pre-labeled andautoclaved 1 mL lyophilization vials. Vials were stoppered with sterilebutyl stoppers and crimped with aluminum caps. All vials were thentransferred to thermal stability ovens. Samples were subject to twothermal stability regimes: (1) two weeks at 40° C. and (2) four weeks at25° C. Throughout this specification, these two temperature regimes aredenoted “T₂” and T₄,” respectively.

B. Size Exclusion Chromatography (SEC)

Etanercept formulations disclosed herein were analyzed using the wellknown technique of Size Exclusion Chromatography (SEC), ahigh-performance liquid chromatography method in which analytes areseparated by size (see Rogner, M. (2000). Size Exclusion Chromatography.Protein Liquid Chromatography. M. Kastner. Amsterdam, Elsevier. 61:89-145.). In order to evaluate thermal stability of the Etanerceptsamples described above, the samples were examined by a SEC method basedon the literature (van Maarschalkerweerd, A., G. J. Wolbink, et al.(2011). “Comparison of analytical methods to detect instability ofetanercept during thermal stress testing.” European Journal ofPharmaceutics and Biopharmaceutics 78(2): 213-221.) The mobile phasebuffer was prepared to contain 50 mM sodium phosphate monobasicmonohydrate and 150 mM arginine. The pH was adjusted to 6.5 using 1 MHCl. All separations were performed using a Tosoh TSK-Gel SWxI 6 mm×4 cmguard column (cat. no. 8543) attached linearly to a Tosoh TSK-Gel G4000SWxI 7.8 mm×30 cm (cat. no. 8542). To perform a separation, the columnswere brought to room temperature (23° C.) and equilibrated with mobilephase at a flow rate of 0.5 mL/min. 5 microliters of 50 mg/mL etanerceptformulation were injected onto the column using an autosampler. Theseparation was accomplished over 30 minutes at a flow rate of 0.5mL/minute. Column eluent was monitored at a wavelength of 280 nm duringthis time.

C. Integration of Size Exclusion Chromatography Chromatograms

All integration was performed using Chromeleon software (Dionex). Priorto integration, the SEC chromatogram for a buffer containing noetanercept was subtracted from all chromatograms. All integration wasperformed between retention times of 12 minutes and 26 minutes. Severalparameters were used to define a peak. The minimum area for a detectedpeak was set to 0.05 mAu*min. The two-dimensional sensitivity for peakdetection was set to 0.01 mAu and 75 seconds. Peak shoulders were addedmanually using a manual integration tool. All detected peaks weremanually adjusted in two steps. First, peak baselines (the bottomboundary of the peak) were adjusted to horizontal. Secondly, thevertical positions of the peak baselines were adjusted to that of thechromatogram baseline. The chromatogram baseline value was defined asthe signal in absence of analyte. The signal in absence of analyte wasdefined as the absorbance in mAu at 12 minutes retention time.

D. SEC Fractions of Etanercept Formulations

In the SEC analysis of etanercept formulations described above, threeSEC chromatography fractions were identified and studied. The fractionsthat were analyzed were, in the order of elution from the SEC column:(1) a high molecular weight fraction representing aggregates of theintact etanercept TNFR:FC fusion protein likely assembled vianon-covalent electrostatic attraction among intact etanercept molecules(hereinafter “aggregate(s)” or aggregate(s) content); (2) monomercontent, representing the intact etanercept TNFR:Fc fusion protein(hereinafter referred to as “monomer” of “monomer content”); (3) afraction likely representing one fragment or a population of fragmentsof the etanercept molecule in which one portion of the TNFR:moleculefusion protein has become cleaved from the monomer; in the loss of anarm of the Fabportion of the fusion protein at the hinge region of themolecule. The most common fragment or clipped species, as measured bySEC, is referred to as Fragment 3. In conducting the SEC analysis, itwill be observed that aggregates elute first, followed by monomer,followed by fragment 3.

The following tables shows the relative amounts of Aggregates, Monomerand Fragment 3 determined by SEC analysis as described above.

TABLE 1 SEC ANALYSIS OF MONOMER Formulation No. t₀ t₁ t₂ CommercialEnbrel 98.81 92.58 87.64 (comparative) [1:2] xx2:6 98.09 — 87.77 xx2:898.07 — 88.38 xx3:10 98.16 — 89.77 xx3:11 98.32 — 89.87 xx3:12 98.33 —90.92 xx3:13 98.18 — 90.74 Note: Amounts reported Tables I, II and IIIare percentages by weight T₀ = formulation maintained at 5° C. andanalyzed within 24 hours of creation. T₁ = formulation stored for oneweek at 40° C. T₂ = formulation stored for two weeks at 40° C.

TABLE II SEC ANALYSIS OF AGGREGATES Formulation No. t₀ t₁ t₂ CommercialEnbrel 0.09 0.59 1.02 (comparative) 2:6 0.30 — 1.81 2:8 0.30 — 1.42 3:100.27 — 1.33 3:11 0.20 — 1.24 3:12 0.21 — 0.85 3:13 0.28 — 0.86 Note:Amounts reported Tables I, II and III are percentages by weight T₀ =formulation maintained at 5° C. and analyzed within 24 hours ofcreation. T₁ = formulation stored for one week at 40° C. T₂ =formulation stored for two weeks at 40° C.

TABLE III ANALYSIS OF FRAGMENT 3 Formulation No t₀ t₁ t₂ CommercialEnbrel 0.00 3.30 6.29 (comparative) 2:6 0.00 — 4.73 2:8 0.00 — 6.29 3:100 3.76 3:11 0 3.59 3:12 0 3.68 3:13 0 3.88

TABLE IV SEC MONOMER CONTENT (4 weeks/25° C.) Table IV below showsmonomer (etanercept) content of etanercept formulations preparedaccording to the present invention, when stored for four weeks at 25 C.°- denoted by the symbol T₄. In the following table T₀ represents SECmeasurements conducted within 24 hours of formulation preparation, atsample temperature of 5° C.; and T₄ represents etanercept formulationsamples subjected to SEC analysis after 4 weeks storage at 25° C. T₀ T₄FORMULATION Monomer Monomer No. Content Content Commercial Enbrel 98.1597.86 (comparative) 3:10 98.16 97.66 3:11 98.32 97.75 3:12 98.33 97.903:13 98.18 97.78

TABLE V SEC AGGREGATES CONTENT (4 weeks/25° C.) Table V below showsaggregate(s) content of etanercept formulations prepared according tothe present invention after storage for four weeks at 25 C. ° . In thefollowing table T₀ represents SEC measurements conducted within 24 hoursof formulation preparation, at sample temperature of 5° C.; and T₄represents etanercept formulation samples subjected to SEC analysisafter 4 weeks storage at 25° C. T₀ T₄ FORMULATION Aggregate(s)Aggregate(s) No. Content Content Commercial Enbrel 0.28 0.25(comparative) 3:10 0.27 0.32 3:11 0.20 0.27 3:12 0.21 0.26 3:13 0.280.32

HIC Analysis of Etanercept Formulations

The following tables (Tables VI and VII) show the results of hydrophobicinteraction chromatography (“HIC chromatography”) conducted on samples3:5 and 3:8. HIC chromatography was carried out in a manner known in theart and generally described in U.S. Pat. No. 7,294,481, incorporatedherein by reference. Samples were evaluated at t₀ (within 24 hours ofpreparation at 5° C.) and again after either two weeks of storage at 25°C. (t₂) (see Table VI) or after 4 weeks of storage at 25° C. (t₄) (SeeTable VII) Peak 1 in the HIC chromatogram is believed to be or include“Fragment 3”, which is identified and quantified using SEC, asreferenced above in the discussion of SEC data; Peak 2 is etanerceptmonomer as referenced above in the discussion of SEC data; and Peak 3includes “Aggregate(s)” as referenced above in the discussion of SECdata. It should further be understood that the terms “peak 1”, “peak 2”and “peak 3 as used here also constitute a reference to the HIC peak 1,peak 2 and peak 3 referred to and disclosed in FIG. 4 of U.S. Pat. No.7,294,481 incorporated herein by reference.

TABLE VI HIC Data after Two Weeks Storage at 40° C. PEAK 1 PEAK 2 PEAK 3Form. # T₀ T₂ T₀ T₂ T₀ T₂ Commercial 0.91 3.23 86.72 83.41 12.33 13.36Enbrel (comparative) 3:10 0.74 3.06 87.36 84.24 11.90 12.70 3:11 0.563.10 86.46 83.73 12.98 13.18 3:12 0.68 3.07 86.80 83.52 12.52 13.40 3:130.77 2.86 86.45 84.33 12.78 12.82

TABLE VII HIC Data after Storage at 25° C. for 4 Weeks PEAK 1 PEAK 2PEAK 3 Form. # T₀ T₄ T₀ T₄ T₀ T₄ Commercial 0.91 1.09 86.76 86.95 12.3311.97 Enbrel (comparative) 3:10 0.74 1.03 87.36 85.83 11.90 13.14 3:110.56 1.11 86.46 85.32 12.98 13.57 3:12 0.68 0.81 86.80 86.36 12.52 12.833:13 0.77 1.01 86.45 85.78 12.78 13.21Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. An aqueous etanercept formulation stabilized toreduce instability, aggregation misfolding and/or fragmentation of theetanercept, said formulation comprising about 25 to about 75 mg/ml ofetanercept and one or more stabilizers, wherein the stabilizers areselected from the group consisting of (i) sodium chloride and (ii)sodium chloride in combination with sucrose or trehalose; and (iii) acombination of sodium chloride, sucrose and trehalose.
 2. Theformulation of claim 1, further comprising one or more additionalcomponents selected from: of a buffer, a tonicity modifier and anexcipient.
 3. The formulation of claim 2 wherein the stabilizer consistsessentially of either: (a) sodium chloride; or (b) sodium chloride incombination with sucrose; or (c) sodium chloride in combination withtrehalose; and wherein, the formulation, optionally, is free oressentially free of arginine, and the formulation elicits long termstorage stability as characterized by at least one of: SEC analysis atM₃ or T₂ or T₄ of: monomer content greater than about 90%; aggregatescontent of less than about 3 wt %; and fragment 3 content less thanabout 5 wt %: and HIC analysis at M₃ or T₂ or T₄ wherein the amount ofthe composition represented by peak 1 of the HIC chromatogram is lessthan about 3 wt. %; the amount of the composition represented by peak 2of the HIC chromatogram is greater than 80 wt. %; and the amount of thecomposition represented by peak 3 of the HIC chromatogram is less thanabout 20 wt. %.
 4. The formulation of claim 2 comprising about 25 to 75mg/ml etanercept, sodium chloride in an amount up to about 150 mM, about1 to about 30 mM sodium phosphate; and about 0 to 5 wt. % sucrose ortrehalose or combination thereof; wherein the composition has a pH ofabout 6.0 to about 6.6; and wherein the composition is characterized bySEC analysis at M₃ or T₂ or T₄ in which: monomer content is greater thanabout 80 wt. %; aggregate(s) content is less than about 3 wt. %, andfragment 3 content is less than about 8 wt. %.
 5. The formulation ofclaim 4, optionally essentially free of arginine, eliciting long termstorage stability characterized by: (a) an SEC analysis at M₃ or T₂ orT₄ of greater than about 90 wt. % monomer content; and less than about 3wt. % aggregate(s) content; and (b) an HIC analysis at M₃ or T₂ or T₄wherein the amount of the composition represented by peak 1 of the HICchromatogram is less than about 4 wt. %; the amount of the compositionrepresented by peak 2 of the HIC chromatogram is greater than about 80wt. %; and the amount of the composition represented by peak 3 of theHIC chromatogram is less than about 20 wt. %; and
 6. The formulation ofclaim 5 having an HIC analysis at M₃ or T₂ or T₄ wherein the amount ofthe composition represented by peak 1 of the HIC chromatogram is lessthan about 1 wt. %; the amount of the composition represented by peak 2of the HIC chromatogram is greater than about 95 wt. %; and the amountof the composition represented by peak 3 of the HIC chromatogram is lessthan about 3 wt. %.
 7. The formulation of claim 2 comprising up to about150 mM sodium chloride, about 1 to 30 mM sodium phosphate, and about 0-5wt. % sucrose or trehalose, or combination of sucrose and trehalose, andhaving a pH of about 6.0 to 6.6; and characterized by: an SEC analysisat M₃ or T₂ or T₄ of greater than about 95 wt. % monomer content andless than about 1 wt. % aggregate(s) content; and an HIC analysis at M₃or T₂ or T₄ wherein the amount of the composition represented by peak 1of the HIC chromatogram is less than or equal to about 3 wt. %; theamount of the composition represented by peak 2 of the HIC chromatogramis greater than about 82 wt. %; and the amount of the compositionrepresented by peak 3 of the HIC chromatogram is less than about 15 wt.%.
 8. The formulation of claim 3 comprising: about 50 mg/ml ofetanercept; about 75 mM sodium chloride; about 10 to 30 mM sodiumphosphate; about 3 wt. % sucrose.
 9. The formulation of claim 3comprising: about 50 mg/ml of etanercept; about 75 mM sodium chloride;about 10 to 30 mM sodium phosphate; about 3 wt. % trehalose.
 10. Theformulation of claim 3 comprising: about 50 mg/ml of etanercept; about75 mM sodium chloride; about 10 to 30 mM sodium phosphate; about 3 wt. %sucrose and having pH of about 6.3 to 6.5.
 11. The formulation of claim2 comprising: about 50 mg/ml of etanercept; about 120 mM sodiumchloride; about 10 to 30 mM sodium phosphate; about 1 wt. % sucrose;about 5 mM arginine, and having pH of about 6.3 to 6.5.
 12. Theformulation of claim 3 comprising: about 50 mg/ml of etanercept; about150 mM sodium chloride; about 10 to 30 mM sodium phosphate, in theabsence of sucrose or trehalose.
 13. The formulation of claim 3comprising: about 50 mg/ml of etanercept; about 100 mM sodium chloride;about 10 to 30 mM sodium phosphate; and about 2 wt. % sucrose.
 14. Theformulation of claim 3 eliciting long term storage stability ascharacterized by: an HIC analysis at M₃ or T₂ or T₄ wherein the amountof the composition represented by peak 2 of the HIC chromatogram isgreater than or equal to about 95 wt. %; and wherein, if peak 3 ispresent on the HIC chromatogram, the amount of the compositionrepresented by peak 3 is less than or equal to about 1 wt. %.
 15. Theformulation of claim 3 having, at M₃ or T₂ or T₄ no more than, onaverage, about 10,000 subvisible particles per mL having a size greaterthan 5 μm.
 16. The formulation of claim 1, containing no arginine, oressentially free of arginine, wherein the composition, at M₃ or T₂ orT₄, elicits long term storage stability that meets one or both of thefollowing criteria: (A) stability comparable to or better thancommercially available etanercept marketed under the trademark Enbrel®,as measured by (i) SEC analysis of the amounts of aggregate(s), monomerand fragment 3 in the composition (as defined in the specification) and(ii) HIC analysis of amounts of material in the compositioncorresponding to peaks 1, 2 and 3 of the HIC chromatogram (as defined inthe specification); and (B) an HIC chromatogram in which (i) peak 3 isabsent, or essentially absent and (ii) peak 2 represents greater thanabout 95 wt % of the composition; an SEC chromatogram containingessentially no peak corresponding to aggregate(s); and an SECchromatogram in which the monomer content represents at least about 95wt % of the composition.